Before the last CASSS Discussion group debate on difference between high temperature and high pressure liquid chromatography started, there was a welcome slide projected on the wall. There was only one sentece (paraphrase):

Meet other people who like and understand what you do

I highlighted the most important part (for me), because I have always problems to explain what I am doing. I would like to ask you all for your thoughts.

• How do you define chromatography?
• Do you have problems to interpret chromatography to other people who don’t understand the chemistry at all?

How do you define chromatography?

In my case, I am always trying to use words as analysing what is inside a sample, separation of complex mixtures, etc. On the very end (when I see that the listener has no clue at all), I am always using examples such as “when you are visiting doctors, they can determine the level of your cholesterol in a blood with chromatography” or “it can be used for a quality control of gasoline in your car”.

Usually, people just answer “ahaa”. And I know, that they still don’t know what I am talking about.

Once I have read the definition of the chromatography as a running race. On the beginning there is a group of a runners and as time flows (mobile phase?;) the group is separated to a groups of the runners with a same speed (retention). On the end of the run, the winner is a non retained compound and the others are individual parts of the mixture. I am not using this expression often, though. But maybe I will.

On the end of the day – as the saying goes – if I am not able to explain what I am doing to my grandparents, then I dont know what I do.

What are your experience and expressions how to define chromatographic separations?

Your comments and suggestions are more than welcome.

I had to solve connection problem in between the Bruker MS and Agilent LC (Agilent shutdown). On the very end, I found out there was no problem in their mutual communication. However, it shows me the future of the instrumental services. WebEx communication.

WebEx is software delivered as a service which you can use it from any computer with an Internet connection. WebEx combines real-time desktop sharing with phone conferencing, so everyone sees the same thing as you talk. And that is exactly what happened.

I asked people from Bruker for advice with my (instrument;) communication problem and we scheduled the WebEx seminar (let’s call it seminar). At exact time I connected to the website they sent me and our online communication started.

For me, it was brand new experience. Ok, I have to say I have no problem with online communication (chat, blogs, social media, …) but it was for first time for me to join the online communiation because of the problem with chromatographic instrument. And I found it very useful.

The online comminication

• saves cost expenses – we all know that it is not always necessary to set up service visit,
• saves time – very important, the seminar can be scheduled on every possible time, which meets requirements of both side
• enhances productivity – majority of the problems can be solve with some kind of advice

I know, all this can be done also using the phone. And for sure, phone is very useful. But using the online communication, you can allow the servicing company the full control over your instrument computer and you don’t have to worry about (almost) anything.

I had this experience with the Bruker company. I am sure others companies offer the same service (or will offer very soon). I belive, that online service is the future of the instrumental service and that majority of the troubleshooting will be solve in this way.

Happy New Chromatographic Year

Not only from the chromatographic point of view, I wish you in the year 2010:

• low pressure
• high efficiency
• sharp resolution
• large capacity
• and satisfactory results

… or (in case you dont want to use chromatographic expressions) you can use any of the translations.

Calibration curve in inverse size-exclusion chromatography (Ve/Vc - elution volume of the polymer divided by the volume of the column, log Mr - logarithm of (polystyrene) molar mass)

The inverse application of the size-exclusion chromatography (SEC) concept, inverse size-exclusion chromatography (ISEC) [1], utilizes a set of molecular probes with defined sizes to determine pore dimensions, and is also referred as chromatographic porosimetry [2].

ISEC provides an alternative to mercury porosimetry or nitrogen adsorption for the determination of the pore size dimensions and the surface area of chromatographic stationary phases. This technique was developed by Halász [3], and has subsequently been extended and refined [4,5,6].

ISEC methodology has been applied to the characterization of a variety of chromatographic stationary phases, including silica [4,5,7,8], silica modified with bonded-phases [9,10] or coated with formamide [11], alumina [8], series of carbohydrate-based size-exlusion gels [6] and synthetic polymer-based adsorbent [12]. The non‑destructive nature of ISEC is and advantage also in structural characterization of monolithic columns [13].

In comparative studies between porosimetry techniques and ISEC, the ISEC method was perceived to require fewer assumptions than mercury porosimetry (e.g., contact angle and surface tension) [5], and to be superior to nitrogen adsorption for following the changes in the surface area, pore volume and pore dimensions that resulted from the grafting of polymeric coatings onto silica [7].

Advantages of inverse size-exclusion chromatography

ISEC has a number of advantages over alternative methods. Column experiments with intact samples packed in a bed can conserve sample integrity and are easy to carry out, as proposed to the special sample preparation procedures in electron microscopy. No other additional equipment other than a chromatography system is necessary for ISEC, so it is relatively inexpensive and convenient.

Operating conditions such as high pressure, low temperature and drying conditions, which are involved in gas sorption or mercury intrusion, are not imposed in ISEC. Experimental conditions similar to those in normal operations result in less significant morphological changes, thus assuring structural information that is relevant to properties of functional interest.

The working pore dimension range of 1 – 400 nm attainable by ISEC [3], which includes resolution not achievable by mercury porosimetry or gas sorption [3,14,15], is of major interest in studies of microporous materials for liquid chromatography.

Limitations

There are number of precautions necessary for realizing effective ISEC procedures. Retention differences are considered to result purely form steric interaction, so solute standards with low polydispersity, i.e., that are well-defined in size and shape, should be used for pore size distribution determination. Dilute standard solutions are typically used to reduce solute-solute interactions, especially aggregation. Appropriate ISEC probes and solvent conditions should be chosen to minimize solute-adsorbent binding and to avoid aggregation.

If these prerequisites for standard ISEC are not satisfied, alternative treatments of non‑standard ISEC must be used to extract the pore size distribution. Potential anomalies include solute adsorption that cannot be eliminated by manipulating solvent conditions and the polydisperse standards when monodisperse solutes are not available. Some adsorbents also contain large pores that are accessible even to the largest polymer standards typically used.

Consequently, the macropore volume cannot be quantitatively differentiated by ISEC, and it is difficult to determine accurately the interstitial volume in a column containing such macroporous media. Micrometer-size latex particles can be used as large probes for quantifying the composition of macropores [16], in this case extra care is needed in choosing the size of the filters and frits in the chromatography system.

References

1. L.G. Aggebrandt, O. Samuelson, J. Appl. Polym. Sci., 8 (1964) 2801.
2. A.A. Gorbunov, L.Y. Solovyova, V.A. Pasechnik, J. Chromatogr., 448 (1988) 307.
3. I. Halász, K. Martin, Angew. Chem. Inter. Ed. (Engl.)., 17 (1978) 901.
4. J.H. Knox, H.P. Scott, J. Chromatogr., 316 (1984) 311.
5. J.H. Knox, H.J. Ritchie, J. Chromatogr., 387 (1987) 65.
6. L. Hagel, M. Ostberg, T. Anderson, J. Chromatogr., 743 (1996) 33.
7. K. Jerabek, A. Revillon, E. Puccillli, Chromatografia, 36 (1993) 259.
8. L.Z. Vilenchik, J.A. Asrar, R.C. Ayotte, L. Ternorutsky, C.J. Hardiman, J. Chromatogr., 648 (1993) 9.
9. I. Mazsaroff, F.E. Regnier, J. Chromatogr., 442 (1988) 15.
10. W.Werner, I. Halász, J. Chromatogr. Sci., 18 (1980) 277.
11. R. Nikolov. W. Werner, I. Halász, J. Chromatogr. Sci., 18 (1980) 207.
12. P. DePhilllips, A.M. Lenhoff, J. Chromatogr. A, 883 (2000) 39.
13. H. Guan, G.Guiochon, J. Chromatogr. A., 731 (1996) 27.
14. A.J. de Vries, M. Lepage, R. Beau, C.I. Guillemi, Anal. Chem., 39 (1967) 935.
15. N.V. Saritha, G. Madras, Chem. Eng. Sci., 56 (2001) 6511.
16. Y. Yao, A.M. Lenhoff, J. Chromatogr. A, 1126 (2006) 107.

Thin layer chromatography is probably the easiest way how to perform chromatographic separation. At least you do not need any instrument. In thin layer chromatography (TLC) the solvent flows through the stationary phase which covers the thin plate. One part of plate is submerged into the mobile phase which travel across the plate using capillary forces.

The sample spots drift towards the second end of the plate according their interaction with the stationary phases. Some of them travel faster then other, hence resulting separation occurs.

The retention factor, Rf, which characterizes the retention of each compound is than calculated as the ratio between the distance of the spot from the beginning and the distance of the solvent front.

One part of thin layer chromatography uses paper as a stationary phase and is accordingly called paper chromatography.

The gas chromatography is special type of chromatography, where the mobile phase is gas, such as helium or nitrogen. The stationary phase is usually solid support covered with liquid layer.

After the sample injection the mobile phase carries the sample compounds through the column. Usually, the temperature gradient is applied and compounds are then separated according theirs boiling points.

Gas chromatography instrumentation

The figure shows typical scheme of gas chromatograph. The gas (mobile phase) flows through the column placed in the oven with controlled temperature. After the separation is finished the individual compounds elute from the column and specific detector registers signal.

Applications

Gas chromatography is very useful for the analysis of small volatile compounds with boiling points lower than 300 °C. Gas chromatography is applied in chemistry industry (especially petrochemistry) to control the quality chemical products. GC can be also used for analysis of toxic compounds, environmental analysis and so on.

Chromatography is analytical chemistry method which is used (and useful) for the separation of complex mixtures of chemical compounds. The main mechanism of the separation is repeatable distribution of the tested compound in between two different phases.

Usually, one phase is solid, fixed in the separation device and the other is moving and flows through the unit. If gas is a second phase, we are referring to the gas chromatography, in case of liquid as a second phase the name is liquid chromatography.

The device where separation takes place is called chromatographic column. This cylindrical shape column is filled with the different kinds of materials – stationary phases. These materials are usually spherical silica particles with different, but well defined, surface chemistry.

The mobile phase flows through the column together with sample (mixture of compounds). Each compound has various affinity to the surface of stationary phase and therefore is separated form each other. In case of ideal state all compounds are eluted from the column in separated bands.

Various techniques are used to recognize these bands and transform them into the signal. In most of the cases the signal draws chromatographic peak – the “hill like” curve describing Gauss distribution.